鸭坦布苏病毒TaqMan荧光定量PCR方法的优化

Optimization of TaqMan-based real-time PCR assay for detection of duck Tembusu virus

为提高鸭坦布苏病毒(DTMUV)TaqMan荧光定量PCR方法的敏感性和简化反应条件,本研究在前期建立的TaqMan荧光定量PCR方法的基础上,设计一条特异性的反转录引物,优化反应体系,建立了简便、快速、敏感的TaqMan荧光定量PCR方法。该荧光定量PCR方法最低检测限为10拷贝,敏感性是未优化的荧光定量PCR方法的5倍、是普通PCR方法的100倍,并且该方法对DTMUV的最低检测限是0.01半数鸡胚致死量(ELD50)。通过批内和批间实验的变异系数表明优化的荧光定量PCR方法的重复性比未优化的荧光定量PCR方法好。通过该优化的荧光定量PCR方法检测其它常见的鸭病病毒的DNAs或RNAs,证明了该方法特异性好。对现地60份疑似DTMUV的样品进行检测,用该方法检出57份样品为阳性,明显高于普通PCR,并且在区分35Ct值左右样品试验中敏感性优于未优化的荧光定量PCR方法。该优化的DTMUV TaqMan荧光定量PCR方法更适用于DTMUV的快速定量流行病学诊断。

To establish a sensitivity method for the detection of the newly emerged duck Tembusu virus （DTMUV）, an optimized TaqMan real-time PCR assay was developed and by using a specific reverse transcription primer and an optimized reaction buffer based previous TaqMan real-time PCR assay. The detection limit of the optimized real-time PCR assay was 10 copies, the sensitivity of this assay was 5 times than non-optimized real-time PCR assay, and sensitive was 100 times than the conventional PCR. For the DTMUV, the detection limit of the optimized assay was 0.01 ELDso. The reproducibility of this optimized real-time PCR assay was verified better than non-optimized assay using inter- and intra- assay. The specificity of the real-time PCR assay were confirmed using RNAs and DNAs extracted fTom other duck viruses. The reliability of this real-time PCR assay was confirmed in 57 of the 60 tissue samples collected from clinical suspected DTMUV infected ducks, the positive coincidence rate was 100% with the conventional PCR and non-optimized real-time PCR. The results reveal that the optimized real-time PCR assay might be a useful diagnostic method for epidemiologically investigating the emerged DTMUV.